Biosensors are devices that use biological components to detect important analytes. Biosensing systems have various applications in areas such as medicine, environmental monitoring, and process control. Classical biosensors are often based on bacteria or purified enzymes that have limitations on efficiency or stability. I have developed several new biosensors to overcome these disadvantages. Two preliminary biosensors were first created based on the extremely strong and specific interaction between biotin and (strept)avidin. Both biosensors showed high sensitivity and reliability for measuring biotin with detection limits of 50-1000 pg/ml and 20-100 ng/ml, respectively. Following these, a new biosensor was developed by coupling a mobile, functionalized microsurface with cell-free expression approaches. This biosensor demonstrated a dynamic range of 1- 100 ng/ml. In addition, I also explored the possibility of combining these biosensing systems with engineered living cells. By leveraging the tools of synthetic biology, a genetic circuit was designed, constructed, and inserted into bacteria for enhanced biotin biosynthesis in vivo. Upon induction, a 17-fold increase in biotin production was measured in the engineered cells in comparison to wild type cells using the biosensors created herein. These new biosensors, particularly the mobile biosensing modality, form a building block for advanced biosensing and drug delivery systems due to enhancements in mobility and specificity. In the future, these biosensing and cellular production systems could impact a range of fields ranging from biomedicine to environmental monitoring. / Master of Science